The present invention relates to a method of determining the prospective benefits of antioxidant supplementation for prevention of cardiovascular disease in diabetic patients, based on polymorphism at the haptoglobin 2 allele.
Cardiovascular disease (CVD) is the most frequent, severe and costly complication of type 2 diabetes.1 It is the leading cause of death among patients with type 2 diabetes regardless of diabetes duration.2 Several population-based studies have consistently shown that the relative risk of CVD in diabetic individuals is several fold higher compared to those without diabetes.3-7 This increased risk appears to be even more striking in women.4,5,8 Risk factors such as hypertension, hyperlipidemia and cigarette smoking all independently increase the relative risk of the diabetic patient of developing CVD, but the effect of diabetes appears to be independent of conventional risk factors.9 
While the incidence of CVD is higher in diabetic patients as compared to non-diabetics in all populations studied, there exist clear geographic and ethnic differences in the relative risk of CVD among diabetic patients that cannot be entirely explained by differences in conventional cardiac risk factors between these groups.10-20 For example, analysis of the relative risk of CVD in different ethnic groups living in the United Kingdom has shown that diabetic patients of South Asian origin have a markedly increased risk12,15, while African-Caribbean diabetic patients have a markedly decreased risk14,16 of CVD as compared to diabetic patients of European origin.
These studies suggest that genetic differences could contribute to differences in susceptibility to CVD in the diabetic patient.
While conceiving the present invention it was hypothesized that a possibility is a functional allelic polymorphism in the haptoglobin gene.
Haptoglobin (Hp) is a hemoglobin-binding serum protein which plays a major role in the protection against heme-driven oxidative stress.23,24 Mice lacking the Hp gene demonstrate a dramatic increase in oxidative stress and oxidative tissue damage particularly in the kidney. In man, there are two common alleles for Hp (1 and 2) manifesting as three major phenotypes 1-1,2-1 and 2-2.21-23 
Functional differences in the hemoglobin-binding capacity of the three phenotypes have been demonstrated. Hp in patients with the Hp 1-1 phenotype is able to bind more hemoglobin on a per gram basis than Hps containing products of the haptoglobin 2 allele.23 Haptoglobin molecules in patients with the haptoglobin 1-1 phenotype are also more efficient antioxidants, since the smaller size of haptoglobin 1-1 facilitates its entry to extravascular sites of oxidative tissue injury compared to products of the haptoglobin 2 allele. This also includes a significantly greater glomerular sieving of haptoglobin in patients with haptoglobin 1-1.22 
The haptoglobin 2 allele appears to have arisen from the 1 allele via a partial gene duplication event approximately 20 million years ago and to have spread in the world population as a result of selective pressures related to resistance to infectious agents.24,25 Presently the haptoglobin alleles differ dramatically in their relative frequency among different ethnic groups.26 The gene duplication event has resulted in a dramatic change in the biophysical and biochemical properties of the haptoglobin protein encoded by each of the 2 alleles. For example, the protein product of the 1 allele appears to be a superior antioxidant compared to that produced by the 2 allele.23 The haptoglobin phenotype of any individual, 1-1, 2-1 or 2-2, is readily determined from 10 microliters of plasma by gel electrophoresis.
It was recently demonstrated that the haptoglobin phenotype is predictive of the development of a number of microvascular complications in the diabetic patient.27-29 Specifically, it was shown that patients who are homozygous for the haptoglobin 1 allele are at decreased risk for developing retinopathy and nephropathy. This effect, at least for nephropathy, has been observed in both type 1 and type 2 diabetic patients and the relevance strengthened by the finding of a gradient effect with respect to the number of haptoglobin 2 alleles and the development of nephropathy.29 Furthermore, it was shown that the haptoglobin phenotype may be predictive of the development of macrovascular complications in the diabetic patient. We have shown that the development of restenosis after percutaneous coronary angioplasty is significantly decreased in diabetic patients with the 1-1 haptoglobin phenotype.27,30 Previous retrospective and cross-sectional studies examining haptoglobin phenotype and coronary artery disease in the general population have yielded conflicting results.31-38 The role of haptoglobin phenotype in the development of atherosclerotic coronary artery disease in the diabetic state has not been studied.
American Indians, previously thought to be resistant to developing coronary artery disease, are presently experiencing CVD in epidemic proportions.20 This increased incidence of CVD has been attributed to the sharp increase in type 2 diabetes in this population.1,2 The Strong Heart Study has examined the incidence, prevalence and risk factors of cardiovascular disease in American Indian populations in three geographic areas since 1988 with continued surveillance to the present.20 The relative genetic homogeneity of this population of patients may permit identification of specific genetic factors that contribute to CVD disease in the diabetic state.
Accordingly, in U.S. Pat. No. 6,613,519, correlation was made, for the first time, for determining the relative risk of CVD in diabetic patients according to haptoglobin phenotype in a case/control sample from the Strong Heart Study.
Some prior art publications teach methods of correlating haptoglobin phenotype and disease. WO98/37419 teaches a method and kit for determining a haptoglobin phenotype and specifically relates to applications involving human haptoglobin. Teachings of this application focus on use of the haptoglobin 2-2 phenotype as an independent risk factor, specifically in relation to target organ damage in refractory essential hypertension, in relation to atherosclerosis (in the general population) and acute myocardial infarction and in relation to mortality from HIV infection. This application does not teach the use of haptoglobin phenotype as a risk factor in cardiovascular disease in DM. Because of the tendency of a haptoglobin 2-2 phenotype to make patients more prone to oxidative stress, it might be argued that use of a 2-2 phenotype as a negative predictor for cardiovascular disease in DM is indirectly implied by this patent. However, teachings of this patent do not include the idea that haptoglobin 1-1 phenotype is a positive predictor for reduced tendency towards cardiovascular disease in DM, or for the effects of antioxidant supplementation. Indeed, in a later study, the authors of PCT WO98/37419 reported opposite results, concluding that Hp 1-1 patients are at elevated risk for cardiovascular disease mortality (De Bacquer et al, Atherosclerosis 2001; 157:161-6). Deriving useful correlations between haptoglobin phenotype and disease requires careful and imaginative analysis, since many studies have reported no or confounding results (Buhlin et al Eur Heart J 2003; 24:2099-107; Lind et al Angiology 2003;54:401-10; Hong et al Hum Hered 1997;47:283-7).
In other words, it has been proposed that oxidative stress originating from Hp 2-1 or 2-2 phenotype leads to vascular complications in the general populations. It is also known that certain vascular complications are associated with oxidative stress associated with DM. At present, however, it remains unclear, and cannot be predicted, whether Hp1-1 phenotype can affect the response to antioxidant supplementation for prevention of vascular complications in diabetic patients.
Teachings of PCT WO98/37419 include use of a haptoglobin binding partner. The binding partner according to PCT WO98/37419 may be any molecule with at least two locations by which it binds haptoglobin. The locations may be formed by a peptide, antibody, or a portion thereof, or by a lectin, a cell receptor, a molecular imprint or a bacterial antigen or a portion thereof. Teachings of this patent focus specifically on the use of the T4 antigen of S. pyogenes. All haptoglobins contain both alpha chains and beta chains. Beta chains are identical in all haptoglobins, while alpha chains differ between the two alleles of the haptoglobin gene. The alpha 2 chain of haptoglobin is the result of a mutation based on an unequal crossing over and includes 142 amino acids, in contrast to the 83 amino acids of the alpha 1 chain. Immunologically the alpha 1 and alpha 2 chains are similar, with the exception of a unique sequence of amino acid residues in the alpha 2 chain (Ala-Val-Gly-Asp-Lys-Leu-Pro-Glu-Cys-Glu-Ala-Asp-Asp-Gly-Gln-Pro-Pro-Pr o-Lys-Cys-Ile, SEQ ID NO:1). Any portion of this unique peptide sequence is therefore a suitable epitope for raising antibodies to differentiate between haptoglobins containing alpha 1 and alpha 2 chains as described in “Using Antibodies: A Laboratory Manual” (Ed Harlow and David Lane eds., Cold Spring Harbor Laboratory Press (1999)) which is fully incorporated herein by reference. Such antibodies might be monoclonal, polyclonal, or any portion thereof and may be enriched or purified by any one of a number of techniques known to those skilled in the art. In addition, the nucleotide sequence encoding this sequence can be readily employed to differentiate among Hp genotypes.
Antioxidants, Haptoglobin and prevention of Cardiovascular Disease (CVD) in Diabetic Patients: The overall prevalence of coronary artery disease is over 55% in adult diabetes mellitus (DM) compared to 2-4% of the general population. Mortality from CVD is more than doubled in men and quadrupled in women who have DM compared with non-diabetics (Stamler, et al. Diabetes Care 1993; 16: 434-444). An increase in oxidative stress represents an attractive unifying mechanism explaining the coordinate activation of several signal transduction pathways known to mediate diabetic vascular disease (Nishikawa et al., Nature 2000; 404:787-790). Hyperglycemia and the oxidative milieu created as a result of glucose autooxidation results in the formation of advanced glycation end-products (AGEs) (Ohgami et al., J Diabetes Complic 2002; 16:56-59) and modified low density lipoproteins (ox-LDL) (Steinberg D J Biol Chem 1997;272:20963-6) which can stimulate the production of multiple inflammatory cytokines implicated in the pathological and morphological changes found in diabetic vascular disease. The oxidation hypothesis is supported by experimental animal data in which antioxidants such as vitamin E have been demonstrated to markedly retard the atherosclerotic process (Williams et al Atherosclerosis 1992; 94: 153-59). However, despite the promising results of in vitro and laboratory studies, several recent, large scale prospective placebo-controlled trials have failed to provide conclusive evidence supporting the benefits of either vitamin E alone (HOPE Study Investigators NE J Med 2000; 342: 154-160; Hodis et al, Circulation 2002; 106:1453-59; Jiang et al, J Biol Chem 2002; 277: 31850-6) nor in combination with other antioxidant vitamins (GISSI, Lancet 1999; 354:4477-55; Brown et al NE J Med 2001;345: 1538-92; Marchioli et al, Lipids; 2001: 36 Suppl:S53-63; Waters et al, JAMA 2002; 288:2432-40; Witztum et al Trends Cardio Med 2001;11:93-102) reduces the incidence of major adverse cardiovascular events. The Heart Outcomes Prevention Evaluation (HOPE) trial was one such study which specifically addressed the efficacy of vitamin E therapy in preventing diabetic CVD (HOPE Study Investigators NE J Med 2000; 342: 154-160). The HOPE study failed to demonstrate any clinical benefit on cardiovascular (CV) outcomes with the daily administration of 400 IU vitamin E for 4.5 years. Several mechanisms have been proposed to explain the apparent failure of vitamin E in these studies. Steinberg has proposed that benefit from antioxidant therapy may only be demonstratable in specific patient subgroups experiencing increased oxidative stress (Steinberg et al Circulation 2002; 105:2107-111).
Vascular complications occur over time in diabetics, even though their blood sugar levels may be controlled by insulin or oral hypoglycaemics (blood glucose lowering) medications. There are a number of vascular complications that diabetics are at risk of developing, including diabetic retinopathy, diabetic cataracts and glaucoma, diabetic nephropathy, diabetic neuropathy, claudication, and gangrene, hyperlipidaemia and cardiovascular problems such as hypertension, atherosclerosis and coronary artery disease. Atherosclerosis may cause angina and heart attacks, and is twice as common in people with diabetes than in those without diabetes, affecting both men and women equally.
A growing body of evidence indicates that such diabetic vascular disease develops only in those patients who are genetically susceptible (UK Prospective Study Group Diab Care 1998;21:1271-77). The haptoglobin gene is polymorphic with two major classes of alleles, denoted 1 and 2. It has been recently demonstrated that this polymorphism in the haptoglobin gene is an independent risk factor for CVD in the diabetic individual (see U.S. Pat. No. 6,613,519, to Levy et al, issued Sep. 2, 2003, Example I hereinbelow, and Levy et al J Am Coll Card 2002; 40: 1984-90). Diabetic patients homozygous for the haptoglobin 2 allele were found to have a 5 fold greater risk of CVD as compared to those homozygous for the haptoglobin 1 allele. The same authors have also demonstrated that the haptoglobin 2 allele protein product is an inferior antioxidant as compared to the haptoglobin 1 allele protein product (Melamed-Frank et al Blood 2001; 98:3693-98). However, the above-mentioned studies neither sought, nor implied, a correlation between antioxidant supplementation and CVD in diabetic patients, and the haptoglobin phenotype, or the usefulness of such a correlation in prediction of benefit to be derived from antioxidant therapy. Therefore, we hypothesized that antioxidant supplementation in diabetic patients homozygous for the haptoglobin 2 allele would be beneficial in preventing adverse cardiovascular events. In order to test this hypothesis we haptoglobin typed participants from the HOPE study and determined the relative risk ratio of major cardiovascular endpoints for the three possible haptoglobin types according to vitamin E and ramipril treatment.
There is a widely recognized need for, and it would be highly advantageous to have a method to predict which specific DM patients have lower risk with respect to cardiovascular disease, and which specific subgroup of patients would benefit from preventative antioxidant therapy. Such a method would allow medical practitioners to make best use of available resources while minimizing risk to each patient to the greatest possible extent.